Secreted key regulators (Fgf1, Bmp4, Gdf3) are expressed by PAC1-immunopositive retinal ganglion cells in the postnatal rat retina.

Identified as a member of the secretin/glucagon/VIP superfamily, pituitary adenylate cyclase-activating polypeptide (PACAP1-38) has been recognized as a hormone, neurohormone, transmitter, trophic factor, and known to be involved in diverse and multiple developmental processes. PACAP1-38 was reported to regulate the production of important morphogens (Fgf1, Bmp4, Gdf3) through PAC1-receptor in the newborn rat retina. To follow up, we aimed to reveal the identity of retinal cells responsible for the production and secretion of Fgf1, Bmp4, and Gdf3 in response to PACAP1-38 treatment. Newborn (P1) rats were treated with 100 pmol PACAP1-38 intravitreally. After 24 h, retinas were dissected and processed for immunohistochemistry performed either on flat-mounted retinas or cryosections. Brn3a and PAC1-R double labeling revealed that 90% of retinal ganglion cells (RGCs) expressed PAC1-receptor. We showed that RGCs were Fgf1, Bmp4, and Gdf3-immunopositive and PAC1-R was co-expressed with each protein. To elucidate if RGCs release these secreted regulators, the key components for vesicle release were examined. No labeling was detected for synaptophysin, Exo70, or NESP55 in RGCs but an intense Rab3a-immunoreactivity was detected in their cell bodies. We found that the vast majority of RGCs are responsive to PACAP, which in turn could have a significant impact on their development or/and physiology. Although Fgf1, Bmp4, and Gdf3 were abundantly expressed in PAC1-positive RGCs, the cells lack synaptophysin and Exo70 in the newborn retina, thus unable to release these proteins. These proteins could regulate postnatal RGC development acting through intracrine pathways.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP), a Novel Secretagogue, Regulates Secreted Morphogens in Newborn Rat Retina.

Purpose: Pituitary adenylate cyclase-activating peptide (PACAP)1-38 has been reported to be responsible for regulation of a disparate array of developmental processes in the central nervous system, and its antiapoptotic effect has been revealed in numerous models, pointing to its relevance in the etiology of neurodegenerative disorders. However, its function in retinal development remains unclear. Here, we aimed to point out that versatility can be achieved through interaction with other regulators, in which PACAP can act indirectly on the retinal microenvironment. Methods: Wistar rats at age postnatal day 1 were injected intravitreally with PACAP or PAC1 receptor antagonist (PACAP6-38, M65) or VPAC1 antagonist (PG97-269) alone or in combination. Retinas were removed at 3, 6, 12, or 24 hours after injection. Changes in mRNA level were assessed using quantitative PCR, whereas changes in protein levels were measured by Western blot. Results: Intravitreal injection of PACAP or PAC1 receptor antagonists or the VPAC1 antagonist showed that PACAP receptors regulate the expression of five key secreted molecules (i.e., Fgf1, Bmp4, Wnt1, Gdf3, and Ihh), wherease other crucial morphogens (i.e., Fgf2, Fgf4, Fgf8, Fgf9, Shh, and Bmp9) were not affected. Pharmacologic dissection revealed that both PAC1 and VPAC1 induced downstream signaling and could cause upregulation of Fgf1, Bmp4, and Wnt1, whereas expression of Gdf3 might be mediated through the VPAC2 receptor. Conclusions: Our data are the first to shed light on PACAP as a secretagogue regulating a sustained production of morphogens, which in turn could enable PACAP to serve as a mitogen for retinal cells, to induce ganglion cell differentiation, and to contribute to RPE development.